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Superb Specific, Ultra Sensitive, and Rapid Identification of Oseltamivir-Resistant H1N1 Virus: Naked-Eye and SERS Dual-Mode Assay using Functional Gold Nanoparticles Gayoung Eom, Ahreum Hwang, Do Kyung Lee, Kyeonghye Guk, Jeong Moon, Jinyoung Jeong, Juyeon Jung, Bongsoo Kim, Eun-Kyung Lim, and Taejoon Kang ACS Appl. Bio Mater., Just Accepted Manuscript • Publication Date (Web): 12 Feb 2019 Downloaded from http://pubs.acs.org on February 12, 2019

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ACS Applied Bio Materials

Superb Specific, Ultra Sensitive, and Rapid Identification of Oseltamivir-Resistant H1N1 Virus: Naked-Eye and SERS Dual-Mode Assay using Functional Gold Nanoparticles Gayoung Eom,†,‡,+ Ahreum Hwang,†,‡ Do Kyung Lee,§ Kyeonghye Guk,†,# Jeong Moon,†,¶ Jinyoung Jeong,#,& Juyeon Jung,†,# Bongsoo Kim,‡ Eun-Kyung Lim,†,#,* and Taejoon Kang†,#,*

†

Bionanotechnology Research Center, KRIBB, Daejeon 34141, Republic of Korea

‡

Department of Chemistry, KAIST, Daejeon 34141, Republic of Korea

§

BioNano Health Guard Research Center, KRIBB, Daejeon 34141, Republic of Korea

#

Department of Nanobiotechnology, KRIBB School of Biotechnology, UST, Daejeon 34113,

Republic of Korea ¶

Department of Chemical and Biomolecular Engineering, KAIST, Daejeon 34141, Republic of

Korea &

Environmental Disease Research Center, KRIBB, Daejeon 34141, Republic of Korea

KEYWORDS Oseltamivir, Influenza Virus, Mutant Virus, Naked-Eye, Surface-enhanced Raman Scattering, Oseltamivir Hexylthiol, Gold Nanoparticle

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ABSTRACT

To prevent the global transmission of mutant viruses and minimize the damage caused by mutant virus infection, the accurate identification of newly emerged mutant viruses should be a priority. The key problem in mutant virus identification is that the selective detection of mutant virus in the biological environment, where small amounts of mutant virus and copious amounts of wild-type virus coexist, is difficult. Herein, we report specific and ultrasensitive detection of oseltamivirresistant (pH1N1/H275Y mutant) virus using functional Au nanoparticles (NPs). The functional Au NPs were prepared by modifying the surfaces of Au NPs with oseltamivir hexylthiol (OHT) and malachite green isothiocyanate (MGITC) simultaneously. OHT is an excellent receptor for the pH1N1/H275Y mutant virus because it has a 250-fold higher binding affinity for the pH1N1/H275Y mutant virus than for the wild-type virus. MGITC is a Raman reporter that provides a distinctive surface-enhanced Raman scattering (SERS) signal. The SERS signal of MGITC on Au NPs makes us to detect pH1N1/H275Y mutant viruses sensitively and quantitatively. The functional Au NPs enable naked-eye and SERS dual-mode detection of mutant viruses. Only in the presence of the pH1N1/H275Y mutant virus, the functional Au NPs aggregate and the color of the NPs changes from red to purple. This allows us to detect mutant viruses with the naked eye. Furthermore, the aggregated Au NPs can provide strong SERS signals of MGITC. By measuring the SERS signals, we could detect the pH1N1/H275Y mutant virus with a detection limit of 10 PFU. Importantly, the pH1N1/H275Y mutant virus could be detected by using the functional Au NPs even in a mixture of mutant and wild-type viruses with a ratio of 1/100. This result suggests that the present method might be employed for the diagnosis of oseltamivirresistant virus and for further research, including mutant virus analysis and drug development.


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1. Introduction The flu is a respiratory (i.e., nose, throat, and lung) infection that can be caused by a variety of influenza viruses. In the United States, influenza viruses cause seasonal epidemics of disease almost every winter. In contrast to the regular seasonal epidemics of influenza, the emergence of new or mutant influenza viruses irregularly causes influenza pandemics. Although the incidence of influenza can vary widely between years, approximately 36,000 deaths and more than 200,000 hospitalizations are directly associated with influenza every year in the United States.1 For the treatment of influenza, there have been many efforts to develop several antiviral drugs. Among them, oseltamivir (Tamiflu) has been most widely used to reduce the symptoms of influenza virus infection.2 Since the approval of oseltamivir by Food and Drug Administration (FDA) in 1999, it has been globally used for the treatment of influenza virus-infected patients. Unfortunately, the wide use of oseltamivir induced the emergence of oseltamivir-resistant (pH1N1/H275Y mutant) viruses first during 2007 ~ 2008 influenza season and the occurrence of pH1N1/H275Y mutant viruses has been sharply increased since then.3,4 Early clinical studies reported that oseltamivir resistance was observed in 1 ~ 2% of adults and 5 ~ 6% of children; however, recent studies revealed that the resistance has increased up to 18% of oseltamivir-treated children.5,6 When the influenza virus-infected patients take oseltamivir, the wild-type viruses in the patients are eliminated, but the oseltamivir-resistant viruses are conserved in the patients.7 Previous studies have reported that the ratio of pH1N1/H275Y mutant viruses to wild-type viruses can be increased from 2.0% to 90.6% after 6 days of oseltamivir treatment and that the complete switch from wildtype to mutant viruses occurs after 9 days of therapy.8,9 Moreover, the patients infected with the oseltamivir-resistant viruses have a higher possibility of developing pneumonia or sinusitis than those infected with wild-type viruses.4,10 In this regard, the Centers for Disease Control and


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Prevention (CDC) and World Health Organization (WHO) announced that pH1N1/H275Y mutant viruses have quickly spread over many countries globally and been a threat to the global public health.11 Typically, influenza viruses have been diagnosed by immunoassays or rapid molecular assays after nasal and throat swab sampling.12-14 These methods are simple and fast; however, it is difficult to identify mutant viruses accurately. Although polymerase chain reaction (PCR)-based genetic sequencing of viruses enables to observe the genetic changes of virus genome, it is timeconsuming and often produce false results due to the amplification of undesired nucleic acid. 15-17 In addition, PCR is still challenging to determine the specific components of mixed virus populations in clinical specimens where a small number of mutant viruses coexist with a large number of wild-type viruses.18 Consequently, the sensitive, selective, and rapid detection method of oseltamivir-resistant influenza viruses is urgently needed to prevent the global transmission of pH1N1/H275Y mutant viruses and to minimize the damage caused by mutant virus infection. Previously, we verified that oseltamivir hexylthiol (OHT) can act as a molecular receptor of the pH1N1/H275Y mutant virus.19 OHT provided 250-fold higher binding affinity for the neuraminidase (NA) of the pH1N1/H275Y mutant virus than that of wild-type virus. This marvelous property of OHT leads us to develop a superb specific and ultra sensitive detection method for mutant viruses by combining OHT with Au nanoparticles (NPs). Au NPs have been a valuable biosensing platform because of their biocompatibility, easy surface modification, high surface-to-volume ratio, and unique optical properties.20-22 They have been used for the development of colorimetric sensors because the color of NPs can be changed by analytes. 23-26 Also, Au NPs have been employed for the development of surface-enhanced Raman scattering (SERS) sensors, as it was reported that a single-molecule detection by using NPs is possible.27-35


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On this basis, herein we developed the functional Au NPs that can detect oseltamivir-resistant virus in naked-eye and SERS dual-mode assays. The functional Au NPs are prepared by modifying the surfaces of Au NPs with OHT and malachite green isothiocyanate (MGITC) simultaneously. The color of the functional Au NPs is red in the neutral state and changes to purple in the presence of the pH1N1/H275Y mutant virus. Based on the color observation with the naked eye, we could detect mutant virus at a low number of 1,000 PFU. Furthermore, 10 PFU of mutant virus was successfully detected by measuring the SERS signals of functional Au NPs. Most importantly, the functional Au NPs could identify the pH1N1/H275Y mutant virus even in the presence of overwhelming amounts of wild-type virus. In the mixture of mutant and wild-type viruses with a ratio of 1/100, strong SERS signals of functional Au NPs were observed. This result indicates that accurate and sensitive diagnosis of the pH1N1/H275Y mutant virus is feasible by using OHTfunctionalized Au NPs. We anticipate that the present method allows the mutant virus-infected patients to receive the maximum therapeutic benefit and contributes to the prevention of mutant virus spread.


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2. Experimental Section Materials and Reagents MGITC was purchased from Setareh Biotech (USA). Glass capillaries were purchased from Marienfeld GmbH & Co (Germany). OHT was synthesized by 4Chem Laboratory (Suwon, Republic of Korea). Sodium citrate and HAuCl4 were purchased from Sigma-Aldrich. Preparation of pH1N1 and pH1N1/H275Y mutant viruses Pandemic H1N1 virus (A/California/07/2009) (pH1N1) was provided by the BioNano Health Guard Research Center (HGUARD) of Korea Research Institute of Bioscience and Biotechnology. The pH1N1/H275Y mutant virus (H275Y mutation A/Korea2785/2009 pdm: NCCP 42017) was obtained from the National Culture Collection for Pathogens (NCCP) operated by the Korea National Institute of Health (KNH). All virus titers were determined by using a one-step RT-PCR kit (Promega) in accordance with the manufacturer’s instructions. Preparation of functional Au NPs For the synthesis of Au NPs, 1 wt % of sodium citrate was dissolved in 100 mL of deionized (DI) water and vigorously stirred at 95°C. Then, 1 mL of a 1 wt % HAuCl4 solution was quickly added to the sodium citrate solution. This mixture was stirred at 95°C until the color of the solution changed from yellow to deep red. The product solution was cooled naturally to room temperature without being disturbed. The prepared Au NP solution (1 mL) was centrifuged at 15,000 rpm for 20 min at 4°C to remove excess sodium citrate and redispersed in DI water. MGITC (20 μL, 1 μM in DI water) and OHT (200 μL, 6 mg/mL in DI water) were added to the Au NP solution and vigorously mixed for 90 min at room temperature. This mixture was centrifuged at 15,000 rpm for 20 min at 4°C and resuspended in DI water. Absorbance spectra were obtained from a UV/VIS spectrometer (Beckman Coulter).


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SERS measurements The pH1N1 virus and pH1N1/H275Y mutant virus were diluted to the desired number of viruses in DI water. The functional Au NPs were incubated with each virus solution for 5 min in a microtube and then collected in a glass capillary tube. SERS measurements were carried out by using a micro-Raman system based on an Olympus BX41 microscope. The excitation source was a He–Ne laser operating at λ=633 nm, and the laser power was 20 mW. The laser spot was focused on the NP solution inside the glass capillary through a 50× objective lens. TEM analysis TEM images were taken on a Tecnai G2 F30 S-Twin microscope operated at 300 kV. The functional Au NP solutions were reacted with pH1N1/H275Y mutant viruses and pH1N1 viruses. After 5 min, the mixture was dropped on a formvar/carbon TEM grid. The viruses are stained with uranyl formate (4% w/v). The grid was dried in air before TEM analysis.


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3. Results and Discussion Figure 1 shows a schematic illustration of the naked-eye and SERS dual-mode sensing procedure for the pH1N1/H275Y mutant virus. Au NP-based colorimetry, which enables the detection of molecules in the naked eye, is simple, fast, and inexpensive. However, this technique often shows unsatisfactory sensitivity.26 SERS is a fascinating phenomenon that significantly increases the Raman signals of molecules near plasmonic nanostructures.36-39 Since SERS has been considered as an ultra-sensitive sensing technique, several Au NP-based SERS assays have been developed.40-43 For the naked-eye and SERS dual-mode sensing of pH1N1/H275Y mutant virus, we prepared the functional Au NPs by modifying the surfaces of Au NPs with OHT and MGITC simultaneously (bottom panel of Figure 1). Specifically, 12 nm Au NPs were mixed with 1 μM MGITC solution and 6 mg/mL OHT solution for 90 min at room temperature. This mixture was then centrifuged at 15,000 rpm for 20 min at 4°C and resuspended in DI water. We synthesized OHT by linking a hexylthiol molecule to the ester groups in oseltamivir phosphate.19 According to a previous result, OHT exhibits 3.3 kcal/mol lower binding free energy in the mutant virus than in the wild-type virus. This binding free energy corresponds to a 250-fold higher binding affinity for the pH1N1/H275Y mutant virus than for the wild-type virus. Therefore, OHT can be an efficient anchor for the specific recognition of the pH1N1/H275Y mutant virus. MGITC is a widely used Raman reporter that has strong absorbance peak about 630 nm and provides a distinctive SERS signal.44 Both OHT and MGITC can be attached at the surface of Au NPs through Au-S bonds. The estimated surface densities of OHT and MGITC is 1.97 × 104 and 2.00 × 102 molecules/single Au NP, respectively. For the detection of the mutant virus, the functional Au NPs are simply added to the pH1N1 virus solution. In the presence of the pH1N1/H275Y mutant virus, the OHT units on the Au NPs interact with the NA sites of the mutant viruses, leading to the


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aggregation of the Au NPs. Consequently, the color of the functional Au NPs changes from red to purple, and the SERS signals of the NPs significantly increase. In the absence of mutant viruses, the functional Au NPs remain in a well-dispersed state, providing a red color and weak SERS signals. As shown in Fig. 1, the functional Au NPs exhibit the merits of both SERS and colorimetry including high sensitivity and easy operation. To confirm the successful preparation of functional Au NPs, we investigated the surface plasmon resonance peak and SERS signals of the as-prepared functional Au NPs. Figure 2a shows the absorbance spectra of bare Au NPs (blue curve) and functional Au NPs (orange curve). The maximum peak of the bare NPs appeared at 520 nm, and that of the functional Au NPs appeared at 525 nm. This redshift suggests that the surfaces of the NPs were modified by MGITC and OHT.45 The absorbance of OHT in DI water exhibited indistinct peak (Figure S1). In addition, we measured the SERS spectrum of the functional Au NPs. For the SERS measurement, as-prepared functional Au NPs were drop-evaporated on a Si substrate and illuminated with a 633 nm excitation source through a 50× objective lens. As shown in Figure 2b, we obtained strong SERS signals from MGITC on the functional Au NPs, indicating that the surfaces of NPs were modified with MGITC. The peaks are identical to the Raman spectrum of MGITC (Figure S2). The optical characteristics of the functional Au NPs allow us to detect pH1N1/H275Y mutant viruses in nakedeye and SERS dual-mode sensing assays. Next, we sought to detect pH1N1/H275Y mutant viruses by using the functional Au NPs. After the preparation of pH1N1/H275Y mutant virus (1,000 PFU) and pH1N1 wild-type virus (1,000 PFU) samples, we simply mixed the functional Au NPs with the mutant and wild-type virus samples separately. Figure 3a shows transmission electron microscope (TEM) images of functional Au NPs after mixing with wild-type viruses (magenta box) and mutant viruses (blue


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box). For TEM analysis, the viruses were stained with 4% w/v uranyl formate. Uranyl cations (UO2+) bind to proteins and sialic acid molecules of the virus, producing a high electron charge density and thus high image contrast.46 Both wild-type and mutant viruses with ~ 100 nm size are clearly shown in the TEM images (marked by arrows), which is in good agreement with previously results reported for the H1N1 influenza virus.47 Importantly, the functional Au NPs are bound to only the pH1N1/H275Y mutant viruses. None of the functional Au NPs interact with the pH1N1 virus, and unbound Au NPs were observed. This result is attributed to the strong binding affinity between OHT and NA sites of pH1N1/H275Y viruses and clearly confirmed the ability of the functional Au NPs to sense pH1N1/H275Y mutant viruses. Figure 3b exhibits the absorbance spectra of functional Au NPs after mixing with pH1N1 wild-type viruses (magenta curve) and pH1N1/H275Y mutant viruses (blue curve). For the pH1N1 virus sample, a 525 nm absorbance peak was observed. This peak is the same as that of the functional Au NPs before mixing with the viruses, suggesting that the functional Au NPs did not interact with the wild-type viruses. For the pH1N1/H275Y sample, the absorbance peak was shifted to 588 nm. This redshift is because the functional Au NPs become very close to each other after the interaction between OHT and NA of the pH1N1/H275U mutant viruses. The absorbance peak shift of 63 nm is enough to detect mutant viruses with the naked eye. For the assessment of SERS-based mutant virus sensing, we obtained SERS spectra of the functional Au NPs in a glass capillary after mixing with pH1N1/H275Y virus (blue spectrum) and pH1N1 wild-type virus (magenta spectrum) samples separately. As shown in Figure 3c, strong SERS signals of MGITC were observed in the presence of pH1N1/H275Y mutant viruses, while weak SERS signals were observed in the presence of pH1N1 wild-type viruses. This SERS result agrees well with the TEM and absorbance results. Overall, the present Au NPs


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modified by OHT and MGITC enable the highly specific detection of the pH1N1/H275Y mutant virus in naked-eye and SERS dual-mode assays. We explored the quantitative analysis of pH1N1/H275Y mutant viruses by using the functional Au NPs. In naked-eye detection mode, the functional Au NPs were mixed with the the color changes in the mixtures were investigated. In addition, the Au NPs were treated with the wild-type viruses (10, 100, and 1,000 PFU) and a blank sample. The blank sample was prepared by replacing the virus solution with the equivalent volume of DI water. Figure 4a displays photographs of functional Au NP solutions after treatment with pH1N1/H275Y viruses, pH1N1 viruses, and the blank sample. The photographs indicate that the color of the functional Au NPs changed from red to purple in the presence of 1,000 PFU of mutant viruses and that the color changes in the wild-type virus solutions were negligible. In SERS detection mode, a mixture of functional Au NPs and influenza viruses was collected in a glass capillary, and a laser was focused on the glass capillary. Figure 4b exhibits the plot of the 1,616 cm-1 band intensity of MGITC versus the influenza virus number. The SERS intensity gradually increased as the number of pH1N1/H275Y mutant viruses increased from 0 to 1,000 PFU. In contrast, the SERS intensities obtained from the pH1N1 virus samples (10, 100, and 1,000 PFU) were comparable to those from the blank sample. The corresponding full SERS spectra also show that the functional Au NP-based SERS method can detect the pH1N1/H275Y mutant viruses quantitatively (Figure 4c,d). We estimated the limit of detection (LOD) in SERS detection mode to be 10 PFU. These dual-mode sensing results clearly verify that the functional Au NPs can specifically recognize pH1N1/H275Y viruses. In naked-eye detection mode, 1,000 PFU of pH1N1/H275Y viruses can be detected after the simple mixing of the functional Au NPs. In SERS mode, the LOD was lower to 10 PFU, and quantitative detection is possible. Notably, the functional Au NPs also can detect mutant viruses


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rapidly. After mixing the functional NPs and pH1N1/H275Y mutant viruses, the color changes immediately. Furthermore, we reacted the functional Au NPs with influenza viruses for only 5 min and directly measured the SERS signals. Even including the SERS measurement time, the mutant viruses can be detected in 10 min. This rapid sensing ability of the functional Au NPs would make the point-of-care diagnosis of patients infected with the pH1N1/H275Y mutant virus possible. We anticipate that the present dual-mode sensing method of pH1N1/H275Y mutant viruses will be selectively used depending on the purpose and the circumstance. For the accurate identification of mutant viruses, the ability to detect mutant viruses specifically from a sample containing small amounts of mutant viruses and copious amounts of wild-type viruses is vital. We investigated the selective detection of pH1N1/H275Y mutant viruses in a mixture of mutant and wild-type viruses. In the mixture samples, the number of pH1N1/H275Y viruses was varied at 0, 10, 100 and 1,000 PFU, and the number of pH1N1 viruses was fixed at 1,000 PFU. Figure 5a shows photographs of functional Au NPs after mixing with the mutant and wild-type virus-coexisting samples. When the sample contained 1,000 PFU of pH1N1/H275Y viruses, the color of functional Au NPs changed to purple. When the samples had 0 ~ 100 PFU of mutant viruses, the color change was not observed with the naked eye. Furthermore, we measured the SERS signals of the functional Au NPs after incubating the NPs with mixtures of mutant and wild-type viruses together (Figure 5b). The SERS intensity of the 1,616 cm-1 band increased as the number of pH1N1/H275Y mutant viruses increased from 0 to 1,000 PFU (Figure 5c). Surprisingly, the functional Au NPs could detect 1% pH1N1/H275Y mutant viruses in the mutant (10 PFU) and wild-type (1,000 PFU) virus-coexisting mixture. In previous studies, detection of less than 5% pH1N1/H275Y mutant viruses in a mixture by using PCR has been reported.48,49 In the present study, we accurately identified pH1N1/H275Y mutant


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viruses in the sample including only 1% mutant viruses and 99% wild-type viruses by using the functional Au NPs. Notably, the present method allows us to routinely detect pH1N1/H275Y mutant viruses with comparable sensitivity to PCR. Considering that the relative population of pH1N1/H275Y viruses is ~ 2.0% at the initial stage of mutation, the functional Au NPs can be utilized for the early diagnosis of mutant viruses. We anticipate that the functional Au NP-based naked-eye and SERS dual-mode sensing technique might be employed to identify the mutant viruses in clinical samples soon.


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4. Conclusion We developed functional Au NPs for the highly specific and ultrasensitive detection of pH1N1/H275Y mutant viruses through naked-eye and SERS dual-mode methods. The color of the functional Au NPs changes from red to purple in the presence of pH1N1/H275Y mutant viruses, allowing us to detect the mutant viruses at a low number of 1,000 PFU with the naked eye. Additionally, we could lower the LOD to 10 PFU by using the SERS detection mode of the functional Au NPs. Furthermore, the functional Au NPs could identify the pH1N1/H275Y mutant virus even in the presence of copious amounts of the wild-type virus. In a mixture consisting of 1% of mutant and 99% of wild-type viruses, we successfully identified the pH1N1/H275Y mutant viruses by using the functional Au NPs. The present method might be employed for the early diagnosis of oseltamivir-resistant viruses and thus contribute to the prevention of mutant virus spread.


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ASSOCIATED CONTENT Supporting Information The following files are available free of charge. Absorbance spectrum of OHT and Raman spectrum of MGITC (PDF) AUTHOR INFORMATION Corresponding Author *E-mail: [email protected] (T.K.) *E-mail: [email protected] (E.–K.L.) Present Addresses +

School of Electrical Engineering, KAIST, Daejeon 34141, Republic of Korea

ACKNOWLEDGMENT This research was supported by the Center for BioNano Health-Guard funded by the Ministry of Science and ICT (MSIT) of Korea as Global Frontier Project (H-GUARD_2013M3A6B2078950 and H-GUARD_2014M3A6B2060507), the Bio & Medical Technology Development Program of the National Research Foundation (NRF) funded by MSIT of Korea (NRF-2018M3A9E2022821), the First-Mover Program for Accelerating Disruptive Technology Development through the NRF funded by MSIT of Korea (NRF-2018M3C1B9069834), the Basic Science Research Program of the NRF funded my MSIT of Korea (NRF-2018R1C1B6005424), and KRIBB initiative Research Program.


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Figures

Figure 1. Schematic illustration of naked-eye and SERS dual-mode sensing procedure for pH1N1/H275Y mutant virus by using functional Au NPs. The functional Au NPs are prepared by modifying the surfaces of Au NPs with OHT and MGITC (bottom panel). In the presence of pH1N1/H275Y mutant viruses, the color of functional Au NPs changes from red to purple, and SERS signals of NPs significantly increase. In the absence of mutant viruses, the functional Au NPs exhibit red color and weak signals.


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Figure 2. (a) Absorbance spectra of bare Au NPs (blue curve) and functional Au NPs (orange curve). The surface plasmon resonance peaks were obtained at 520 nm for bare Au NPs and at 525 nm for functional Au NPs. The number densities of particles were calculated to be 7.46 × 1029 particles/mL for bare Au NPs and 7.43 × 1029 particles/mL for functional Au NPs. (b) SERS spectra of MGITC measured from functional Au NPs.


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Figure 3. (a) TEM images of functional Au NPs after mixing with pH1N1 wild-type viruses (magenta box) and pH1N1/H275Y mutant viruses (blue box). The viruses with ~ 100 nm size are marked by arrows. Scale bars denote 50 nm. (b) Absorbance spectra of functional Au NPs after


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mixing with pH1N1 wild-type viruses (magenta curve) and pH1N1/H275Y mutant viruses (blue curve). The surface plasmon resonance peaks are obtained at 525 nm for pH1N1 wild-type virus and at 588 nm for pH1N1/H275Y mutant virus. The number of influenza viruses is 1,000 PFU. (c) SERS spectra of functional Au NPs in a glass capillary after mixing with pH1N1 wild-type viruses (magenta spectra) and pH1N1/H275Y mutant viruses (blue spectra). The number of influenza viruses is 1,000 PFU.


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Figure 4. (a) Photos of functional Au NPs after mixing with pH1N1/H275Y mutant viruses (10, 100, 1,000 PFU), pH1N1 wild-type viruses (10, 100, 1,000 PFU), and no virus (0 PFU). Blank sample is prepared by replacing virus solution with the equivalent volume of DI water. (b) Plot of 1,616 cm-1 band intensity of MGITC versus the virus number. Data represent the average plus/minus standard deviation from 5 measurements. (c,d) SERS spectra of functional Au NPs in a glass capillary after mixing with pH1N1/H275Y mutant viruses (0, 10, 100, 1,000 PFU) and pH1N1 wild-type viruses (0, 10, 100, 1,000 PFU).


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Figure 5. (a) Photos of functional Au NPs after incubation with the mixture of pH1N1/H275Y mutant and pH1N1 wild-type viruses. The mixed virus solutions are prepared by mixing the


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desired number of pH1N1/H275Y mutant viruses (0, 10, 100, 1,000 PFU) and the fixed number of pH1N1 wild-type viruses (1,000 PFU). The sample with 0 PFU of pH1N1/H275Y mutant virus is prepared by replacing the virus solution with the equivalent volume of DI water. (b) SERS spectra of functional Au NPs in a glass capillary after mixing with the mixture of pH1N1/H275Y mutant (0, 10, 100, 1,000 PFU) and pH1N1 wild-type viruses (1,000 PFU). (c) Intensities of MGITC band at 1,616 cm-1 band measured from the functional Au NPs after incubating with the mixture of pH1N1/H275Y mutant and pH1N1 wild-type viruses. Data represent the average plus/minus standard deviation from 5 measurements.


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Superb Specific, Ultra Sensitive, and Rapid Identification of

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